differential effects of whole red raspberry polyphenols
TRANSCRIPT
University of Massachusetts Amherst University of Massachusetts Amherst
ScholarWorks@UMass Amherst ScholarWorks@UMass Amherst
Nutrition Department Faculty Publication Series Nutrition
2021
Differential Effects of Whole Red Raspberry Polyphenols and Their Differential Effects of Whole Red Raspberry Polyphenols and Their
Gut Metabolite Urolithin A on Neuroinflammation in BV-2 Gut Metabolite Urolithin A on Neuroinflammation in BV-2
Microglia Microglia
Ashley Mulcahy Toney
Mahaa Albusharif
Duncan Works
Luke Polenz
Stacie Schlange
See next page for additional authors
Follow this and additional works at: https://scholarworks.umass.edu/nutrition_faculty_pubs
Authors Authors Ashley Mulcahy Toney, Mahaa Albusharif, Duncan Works, Luke Polenz, Stacie Schlange, Virginia Chaidez, Amanda E. Ramer-Tait, and Soonkyu Chung
International Journal of
Environmental Research
and Public Health
Article
Differential Effects of Whole Red Raspberry Polyphenols andTheir Gut Metabolite Urolithin A on Neuroinflammation inBV-2 Microglia
Ashley Mulcahy Toney 1, Mahaa Albusharif 2, Duncan Works 1,2, Luke Polenz 1, Stacie Schlange 1,Virginia Chaidez 1, Amanda E. Ramer-Tait 2,3 and Soonkyu Chung 1,4,*
�����������������
Citation: Toney, A.M.; Albusharif, M.;
Works, D.; Polenz, L.; Schlange, S.;
Chaidez, V.; Ramer-Tait, A.E.;
Chung, S. Differential Effects of Whole
Red Raspberry Polyphenols and Their
Gut Metabolite Urolithin A on
Neuroinflammation in BV-2 Microglia.
Int. J. Environ. Res. Public Health 2021,
18, 68. https://dx.doi.org/10.3390/
ijerph18010068
Received: 30 November 2020
Accepted: 20 December 2020
Published: 24 December 2020
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional claims
in published maps and institutional
affiliations.
Copyright: © 2020 by the authors. Li-
censee MDPI, Basel, Switzerland. This
article is an open access article distributed
under the terms and conditions of the
Creative Commons Attribution (CC BY)
license (https://creativecommons.org/
licenses/by/4.0/).
1 Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA;[email protected] (A.M.T.); [email protected] (D.W.); [email protected] (L.P.);[email protected] (S.S.); [email protected] (V.C.)
2 Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE 68588, USA;[email protected] (M.A.); [email protected] (A.E.R.-T.)
3 Nebraska Food for Health Center, University of Nebraska-Lincoln, Lincoln, NE 68588, USA4 Department of Nutrition, University of Massachusetts-Amherst, Amherst, MA 01003, USA* Correspondence: [email protected]
Abstract: Whole red raspberry polyphenols (RRW), including ellagic acid, and their gut-derivedmetabolite, urolithin A (UroA), attenuate inflammation and confer health benefits. Although re-sults from recent studies indicate that polyphenols and UroA also provide neuroprotective effects,these compounds differ in their bioavailability and may, therefore, have unique effects on limitingneuroinflammation. Accordingly, we aimed to compare the neuroprotective effects of RRW andUroA on BV-2 microglia under both 3 h and 12 and 24 h inflammatory conditions. In inflammationinduced by lipopolysaccharide (LPS) and ATP stimulation after 3 h, RRW and UroA suppressed pro-inflammatory cytokine gene expression and regulated the JNK/c-Jun signaling pathway. UroA alsoreduced inducible nitric oxide synthase gene expression and promoted M2 microglial polarization.During inflammatory conditions induced by either 12 or 24 h stimulation with LPS, UroA—but notRRW—dampened pro-inflammatory cytokine gene expression and suppressed JNK/c-Jun signaling.Taken together, these results demonstrate that RRW and its gut-derived metabolite UroA differentiallyregulate neuroprotective responses in microglia during 3 h versus 12 and 24 h inflammatory conditions.
Keywords: red raspberry polyphenols; urolithin A; ellagic acid; neuroinflammation; microglia;inflammation; JNK
1. Introduction
The incidence of neurological disorders and diseases, such as Alzheimer’s disease, isincreasing worldwide [1]. Neuroinflammation drives the development of these diseasesas resident macrophage-like cells, microglia, become chronically activated and induce apro-inflammatory environment in the brain [2]. Microglia survey the surrounding area forsigns of damage to nearby cells or tissues [3]. Under homeostatic conditions, microglia arealternatively activated and promote neuroprotection by adopting an M2 polarization state.However, when microglia detect damage-associated molecular patterns (DAMPs), theybecome classically activated, polarize to an M1 phenotype, and induce a pro-inflammatoryenvironment [3]. Because prolonged oxidative stress and inflammation ultimately result inthe development of neurodegenerative diseases, interventions targeting classical microglialactivation are needed.
Red raspberries have been studied extensively for their anti-inflammatory proper-ties and their ability to attenuate inflammation and oxidative stress in multiple chronicdiseases, including cardiovascular disease, obesity, cancer, and Alzheimer’s disease [4,5].The United States is the third largest red raspberry producer, with an industry valued at
Int. J. Environ. Res. Public Health 2021, 18, 68. https://dx.doi.org/10.3390/ijerph18010068 https://www.mdpi.com/journal/ijerph
Int. J. Environ. Res. Public Health 2021, 18, 68 2 of 11
more than $270 million according to estimates in 2014, suggesting consumption of rasp-berries has a positive impact on berry growers [6]. Moreover, the United States exports anestimated 57.6 million pounds of raspberries to other countries but also imports raspberriesworldwide to meet consumer demands [6]. Raspberries are rich in polyphenols, includinganthocyanins and ellagitannins. Ellagic acid (EA), a free form of ellagitannin (ET), resistshydrolysis in the stomach and subsequently undergoes microbial conversion into urolithinsin the gut [5]. Of these gut-derived metabolites, urolithin A (UroA) has been studied ex-tensively and can achieve bioavailability in human plasma once conjugated by phase IIbiotransformation in the liver [7,8]. Although most polyphenolic compounds have lowbioavailability, increased concentrations of UroA can be detected in the plasma and brainsof rats fed pomegranates rich in ellagic acid, thereby suggesting that UroA can provideneuroprotective effects in vivo by passing through the blood brain barrier [9–11]. Moreover,free UroA has been detected in circulation and peripheral tissues during inflammatoryconditions [12].
Numerous studies have specifically documented the ability of red raspberry polyphe-nols or their gut-derived metabolites, urolithins, to attenuate inflammation and oxidativestress [13–16]. EA, as well as its digested metabolites, have been shown in vitro to protectneuroblastoma cells against oxidative stress [10]. UroA, in particular, is known to decreaseneuroinflammation in both in vitro and in vivo models via regulation of autophagy andSIRT-1 activation [17] and MAPK/Akt/NF-kB signaling pathways [18]. Although stud-ies indicate that c-Jun N-terminal kinase (JNK) signaling promotes pro-inflammatoryresponses in microglia [19,20], its role in UroA-mediated neuroprotection has not beenassessed. Despite these promising results, concerns regarding the bioaccesibility of rasp-berry polyphenols warrants a direct comparison of the beneficial effects of polyphenols andtheir gut metabolite, UroA, in regulating cytokine responses and oxidative stress signalingpathways during both 3 h and 12 and 24 h neuroinflammatory conditions.
The aim of this study, therefore, was to compare the neuroprotective effects of redraspberry polyphenols from whole fruit (RRW) versus UroA in BV-2 microglia cells usingtwo different time windows (3 h versus 12 and 24 h induced inflammation). We reportthat UroA and RRW exhibited differential temporal regulation of pro-inflammatory geneexpression and JNK/c-Jun signaling pathways in BV-2 microglia.
2. Materials and Methods2.1. Whole Red Raspberry Polyphenol Preparation
Freshly frozen “Wakefield” red raspberries were donated by Enfield Farms (Lynden, WA)and were used for this study. Extraction of red raspberry polyphenols from whole fruits(i.e., RRW) was conducted using a protocol previously described by our laboratory [21].Phenolic composition of RRW extracts is found in Table S1. Stock concentrations of RRW(20 mg/mL dissolved in PBS) were stored at −80 ◦C and diluted fresh immediately priorto the start of each experiment.
2.2. Cell Culture
Murine BV-2 microglia were provided by Dr. Janos Zempleni from the Universityof Nebraska, Lincoln. Urolithin A (UroA) was obtained as previously described [22].UroA stock concentrations (10 mM) were prepared in DMSO and freshly diluted usingPBS (HyClone, Marlborough, MA, USA). For all experiments, cells were cultured in HighGlucose Dulbecco’s Minimal Essential Media containing 4.0 mM L-glutamine withoutsodium pyruvate (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) supplementedwith 10% fetal bovine serum (Atlanta Biologicals, Flowery Branch, GA, USA), penicillin(100 IU/mL), and streptomycin (100 µg/mL) (HyClone). Cells were maintained in ahumidified atmosphere at 37 ◦C and 5% CO2. Medium was changed every 36–48 h. BV-2cells were seeded at 4 × 105 cells per 6-well plate for all experiments.
To investigate the role of RRW and UroA in microglial inflammation, BV-2 cells werepretreated with either 10 µg/mL RRW or 10 µM UroA for 12 h. Cells were then subjected
Int. J. Environ. Res. Public Health 2021, 18, 68 3 of 11
to either lipopolysaccharide (LPS, 500 ng/mL) treatment for 3 h followed by ATP (2.5 mM)stimulation for 30 min in Opti-MEM (Gibco) or LPS (500 ng/mL) treatment for 12 or 24 h.Concentrations of RRW polyphenols (10 µg/mL) and UroA (10 µM) were selected becausethey have been previously shown to induce beneficial responses in BV-2 microglia [17]as well as J774 and bone marrow-derived macrophages (BMDM) [21]. To investigate theeffects of RRW and UroA on M2 polarization, BV-2 cells were pre-treated with either RRW(10 µg/mL) or UroA (10 µM) for 12 h and then stimulated with interleukin (IL)-4 and IL-13(Peprotech, Cranbury, NJ, USA) in Opti-MEM for 24 h.
2.3. Total RNA Extraction and RT-qPCR
Total RNA was isolated using the TRIzol Reagent (Invitrogen, Carlsbad, CA, USA). RNAconcentrations were measured using a Synergy HT Microplate Reader (BioTek, Winooski, VT,USA; Gen5 Version 3.08), and 2 µg of mRNA was converted into cDNA in a total volume of20 µL (iScript cDNA Synthesis Kit, Bio-Rad, Hercules, CA, USA). Gene-specific primersfor qRT-PCR (Table S2) were purchased from Integrated DNA Technologies (Coralville,IA, USA). Gene expression was determined by real-time qPCR (Quant Studio 5, AppliedBiosystems, ThermoFisher Scientific, (Waltham, MA, USA) using SYBR green (Thermo Scientific,Waltham, MA, USA). Relative gene expression was determined based on the 2−∆∆CT methodwith normalization of the raw data to hypoxanthine-guanine phosphoribosyltransferase (Hprt).
2.4. Western Blot Analysis
Cell extracts were prepared in RIPA buffer containing protease (Millipore Sigma,Burlington, MA, USA) and phosphatase inhibitors (Millipore Sigma) (2 mM Na3VO4,20 mM β-glycerophosphate, and 10 mM NaF). Total protein concentrations were de-termined using the Pierce BCA Protein Assay Kit (ThermoFisher, Waltham, MA, USA).Cell culture supernatants were collected and centrifuged (10,000× g, 5 min) to removecell debris. Supernatant protein was precipitated using the trichloroacetic acid (TCA)precipitation method. Briefly, one volume of 100% (w/v) TCA stock was added to fourvolumes of supernatant sample, incubated overnight at 4 ◦C, and centrifuged at 15,000× gfor 10 min. Supernatants were removed and the pellet was washed with 200 µL coldacetone at 15,000× g for 10 min. Pellets were dried in a 95 ◦C heat block for 5 min toevaporate remaining acetone and resolved with water and SDS sample loading buffer(LI-COR Biosciences, Lincoln, NE, USA).
Proteins (10 µg for cell extracts and total precipitated cell supernatants) were frac-tionated using 8–12% sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis(PAGE) and transferred to polyvinylidene fluoride low-fluorescent (PVDF) membranes(Millipore Sigma). Membranes were blocked (Odyssey blocking buffer, LI-COR) for 1 hat room temperature (RT), washed with 1 × TBS-T (20 mM Tris-HCl + 150 mM NaCL +0.01% Tween 20), and incubated with primary antibody (Table S3) in blocking buffer at 4 ◦Covernight and then the corresponding secondary antibody (Table S3) in blocking buffercontaining 0.05% Tween-20 and 0.01% SDS at RT for 1 h. Blots were scanned using theOdyssey Infrared Imaging System (LI-COR, Lincoln, NE, USA) and analyzed with ImageStudio Lite Software Ver 5.2.5 (LI-COR, Lincoln, NE, USA). Total protein for each blot wasmeasured using the RevertTM 700 Total Protein Stain Kit (LI-COR, Lincoln, NE, USA) andused for normalization and quantification.
2.5. Statistical Analysis
All data were analyzed using one-way ANOVA followed by Tukey’s multiple com-parison tests * p < 0.05 or ** p < 0.01. All analyses were preformed using Graph Pad Prism(Ver 8.3.1., La Jolla, CA, USA).
Int. J. Environ. Res. Public Health 2021, 18, 68 4 of 11
3. Results3.1. Whole Red Raspberry Polyphenols and Urolithin A Decreased NeuroinflammatoryCytokine Expression
Previous research has reported that LPS and ATP induce an inflammasome responsein this cell line [23]. Although these conditions increased expression of Nlrp3, they did notinduce changes in the expression of other inflammasome construct genes (e.g., Asc and Tlr4),nor did they yield detectable levels of activated caspase-1 in culture supernatants (FigureS1). However, LPS and ATP stimulation significantly upregulated expression of genesencoding for the pro-inflammatory cytokines interleukin-1 beta (Il1b), interleukin-6 (Il6),and tumor necrosis factor alpha (Tnf ) compared to unstimulated cells (Figure 1). Both RRWand UroA significantly decreased expression of Il1b and Il6 compared to untreated cellsstimulated with LPS and ATP (Figure 1). UroA, but not RRW, significantly decreased Tnfgene expression after LPS and ATP treatment (Figure 1). Taken together, these resultssuggest that UroA, the gut-derived metabolite from RRW, and, in part, RRW can protectagainst neuroinflammation induced by LPS and ATP by limiting pro-inflammatory cytokineresponses.
Int. J. Environ. Res. Public Health 2021, 18, x FOR PEER REVIEW 4 of 11
2.5. Statistical Analysis
All data were analyzed using one-way ANOVA followed by Tukey’s multiple com-
parison tests * p < 0.05 or ** p < 0.01. All analyses were preformed using Graph Pad Prism
(Ver 8.3.1., La Jolla, CA, USA).
3. Results
3.1. Whole Red Raspberry Polyphenols and Urolithin A Decreased Neuroinflammatory Cytokine
Expression
Previous research has reported that LPS and ATP induce an inflammasome response
in this cell line [23]. Although these conditions increased expression of Nlrp3, they did not
induce changes in the expression of other inflammasome construct genes (e.g., Asc and
Tlr4), nor did they yield detectable levels of activated caspase-1 in culture supernatants
(Figure S1). However, LPS and ATP stimulation significantly upregulated expression of
genes encoding for the pro-inflammatory cytokines interleukin-1 beta (Il1b), interleukin-6
(Il6), and tumor necrosis factor alpha (Tnf) compared to unstimulated cells (Figure 1). Both
RRW and UroA significantly decreased expression of Il1b and Il6 compared to untreated
cells stimulated with LPS and ATP (Figure 1). UroA, but not RRW, significantly decreased
Tnf gene expression after LPS and ATP treatment (Figure 1). Taken together, these results
suggest that UroA, the gut-derived metabolite from RRW, and, in part, RRW can protect
against neuroinflammation induced by LPS and ATP by limiting pro-inflammatory cyto-
kine responses.
Figure 1. Both whole red raspberry polyphenols and urolithin A decreased pro-inflammatory cy-
tokine gene expression. V-2 cells treated with LPS (500 ng/mL) for 3 h followed by ATP (2.5 mM)
for 30 min with or without whole red raspberry polyphenols (10 µg/mL) or urolithin A (10 µM).
Gene expression levels measured by RT-qPCR for Il1b, Il6, and Tnf. All values are represented as
the mean ± SEM. Treatments with different letters indicate significant differences by one-way
ANOVA, p < 0.05. LPS = lipopolysaccharide; RRW = whole red raspberry polyphenols; UroA =
urolithin A.
3.2. Whole Red Raspberry Polyphenols and Urolithin a Regulated JNK/c-Jun Signaling
Pathways during LPS and ATP-Induced Inflammation
Previous literature has reported that UroA regulates the mitogen-activated protein
kinase (MAPK)/NF-κB pathways to protect against neuroinflammation [18,24], but the
role of the JNK/c-Jun pathway in UroA-mediated protection remains unknown. We there-
fore investigated the effect of RRW and UroA on c-Jun N-terminal kinases (JNK) and sub-
sequent p-c-Jun protein production during LPS and ATP-induced neuroinflammation.
Both p-JNK and p-c-Jun protein expression was increased following treatment with
LPSplus ATP compared to treatment with only LPS (Figure 2), an observation consistent
with previous reports connecting the c-Jun pathway to stress and inflammation in micro-
glia [20]. Importantly, RRW and UroA significantly decreased the phosphorylation of both
p-JNK and p-c-Jun compared to untreated microglia stimulated with LPS and ATP (Figure
2). These results, therefore, suggest that regulation of the JNK/c-Jun pathways may
Figure 1. Both whole red raspberry polyphenols and urolithin A decreased pro-inflammatorycytokine gene expression. V-2 cells treated with LPS (500 ng/mL) for 3 h followed by ATP (2.5 mM)for 30 min with or without whole red raspberry polyphenols (10 µg/mL) or urolithin A (10 µM).Gene expression levels measured by RT-qPCR for Il1b, Il6, and Tnf. All values are represented as themean ± SEM. Treatments with different letters indicate significant differences by one-way ANOVA,p < 0.05. LPS = lipopolysaccharide; RRW = whole red raspberry polyphenols; UroA = urolithin A.
3.2. Whole Red Raspberry Polyphenols and Urolithin a Regulated JNK/c-Jun Signaling Pathwaysduring LPS and ATP-Induced Inflammation
Previous literature has reported that UroA regulates the mitogen-activated proteinkinase (MAPK)/NF-κB pathways to protect against neuroinflammation [18,24], but therole of the JNK/c-Jun pathway in UroA-mediated protection remains unknown. We thereforeinvestigated the effect of RRW and UroA on c-Jun N-terminal kinases (JNK) and subsequentp-c-Jun protein production during LPS and ATP-induced neuroinflammation. Both p-JNK andp-c-Jun protein expression was increased following treatment with LPSplus ATP comparedto treatment with only LPS (Figure 2), an observation consistent with previous reportsconnecting the c-Jun pathway to stress and inflammation in microglia [20]. Importantly, RRWand UroA significantly decreased the phosphorylation of both p-JNK and p-c-Jun comparedto untreated microglia stimulated with LPS and ATP (Figure 2). These results, therefore,suggest that regulation of the JNK/c-Jun pathways may contribute to neuroprotective ef-fects by polyphenols and polyphenol-derived metabolites by limiting microglial activationand stress.
Int. J. Environ. Res. Public Health 2021, 18, 68 5 of 11
Int. J. Environ. Res. Public Health 2021, 18, x FOR PEER REVIEW 5 of 11
contribute to neuroprotective effects by polyphenols and polyphenol-derived metabolites
by limiting microglial activation and stress.
Figure 2. Both whole red raspberry polyphenols and urolithin A attenuated neuroinflammation and regulated JNK/c-Jun
signaling pathways. BV-2 cells treated with LPS (500 ng/mL) for 3 h followed by ATP (2.5 mM) for 30 min with or without
whole red raspberry polyphenols (10 µg/mL) or urolithin A (10 µM). Protein expression from cell lysates for p-JNK, t-JNK,
p-c-Jun, and c-Jun by Western blot analysis. Relative protein intensities normalized to total-JNK or c-Jun by LICOR Image
Studio Lite (right). All values are represented as the mean ± SEM. Treatments with different letters indicate significant
differences by one-way ANOVA, p < 0.05. LPS = lipopolysaccharide; RRW = whole red raspberry polyphenols; UroA =
urolithin A.
3.3. Urolithin A Reduced iNOS Gene Expression and Promoted M2 Microglia Polarization
During 3 h inflammatory responses, low levels of inducible nitric oxide synthase
(iNOS) can protect the central nervous system from injury; however, chronically activated
microglia generate high levels of iNOS, which is detrimental and indicative of neuroin-
flammation [25]. We observed a significant increase in iNos expression after stimulating
BV-2 cells with LPS and ATP (Figure 3A). However, both RRW and UroA significantly
decreased iNos expression (Figure 3A).
Previously, we demonstrated that UroA induces M2 macrophage polarization in
BMDM [26]. We therefore tested whether UroA could similarly induce M2 polarization in
microglia. We observed a significant increase in the expression of the Chitinase-like 3 gene
(Ym1; also known as Chi313) in BV-2 microglia pretreated with UroA compared to control
upon M2 stimulation with IL-4 plus IL-13 alone (Figure 3B). Notably, RRW treatment did
not alter Ym1 expression. Collectively, these results indicate that UroA promotes M2 po-
larization in microglia similar to other macrophage-like cells, which may be a unique
mechanism by which UroA, but not red raspberry polyphenols, mediates neuroprotective
effects.
Figure 3. Urolithin A attenuated inducible nitric oxide synthase expression and promoted M2 mi-
croglial polarization. (A) BV-2 cells treated with LPS (500 ng/mL) for 3 h followed by ATP (2.5
mM) for 30 min with or without whole red raspberry polyphenols (10 µg/mL) or urolithin A (10
µM). Gene expression levels measured by RT-qPCR for iNos. (B) BV-2 cells treated with IL-4 (100
ng/mL) and IL-13 (10 ng/mL) for 24 h with or without whole red raspberry polyphenols (10
µg/mL) or urolithin A (10 µM). Gene expression levels measured by RT-qPCR for Ym1. All values
Figure 2. Both whole red raspberry polyphenols and urolithin A attenuated neuroinflammation and regulated JNK/c-Junsignaling pathways. BV-2 cells treated with LPS (500 ng/mL) for 3 h followed by ATP (2.5 mM) for 30 min with orwithout whole red raspberry polyphenols (10 µg/mL) or urolithin A (10 µM). Protein expression from cell lysates forp-JNK, t-JNK, p-c-Jun, and c-Jun by Western blot analysis. Relative protein intensities normalized to total-JNK or c-Jun byLICOR Image Studio Lite (right). All values are represented as the mean ± SEM. Treatments with different letters indicatesignificant differences by one-way ANOVA, p < 0.05. LPS = lipopolysaccharide; RRW = whole red raspberry polyphenols;UroA = urolithin A.
3.3. Urolithin A Reduced iNOS Gene Expression and Promoted M2 Microglia Polarization
During 3 h inflammatory responses, low levels of inducible nitric oxide synthase(iNOS) can protect the central nervous system from injury; however, chronically activatedmicroglia generate high levels of iNOS, which is detrimental and indicative of neuroinflam-mation [25]. We observed a significant increase in iNos expression after stimulating BV-2cells with LPS and ATP (Figure 3A). However, both RRW and UroA significantly decreasediNos expression (Figure 3A).
Int. J. Environ. Res. Public Health 2021, 18, x FOR PEER REVIEW 5 of 11
contribute to neuroprotective effects by polyphenols and polyphenol-derived metabolites
by limiting microglial activation and stress.
Figure 2. Both whole red raspberry polyphenols and urolithin A attenuated neuroinflammation and regulated JNK/c-Jun
signaling pathways. BV-2 cells treated with LPS (500 ng/mL) for 3 h followed by ATP (2.5 mM) for 30 min with or without
whole red raspberry polyphenols (10 µg/mL) or urolithin A (10 µM). Protein expression from cell lysates for p-JNK, t-JNK,
p-c-Jun, and c-Jun by Western blot analysis. Relative protein intensities normalized to total-JNK or c-Jun by LICOR Image
Studio Lite (right). All values are represented as the mean ± SEM. Treatments with different letters indicate significant
differences by one-way ANOVA, p < 0.05. LPS = lipopolysaccharide; RRW = whole red raspberry polyphenols; UroA =
urolithin A.
3.3. Urolithin A Reduced iNOS Gene Expression and Promoted M2 Microglia Polarization
During 3 h inflammatory responses, low levels of inducible nitric oxide synthase
(iNOS) can protect the central nervous system from injury; however, chronically activated
microglia generate high levels of iNOS, which is detrimental and indicative of neuroin-
flammation [25]. We observed a significant increase in iNos expression after stimulating
BV-2 cells with LPS and ATP (Figure 3A). However, both RRW and UroA significantly
decreased iNos expression (Figure 3A).
Previously, we demonstrated that UroA induces M2 macrophage polarization in
BMDM [26]. We therefore tested whether UroA could similarly induce M2 polarization in
microglia. We observed a significant increase in the expression of the Chitinase-like 3 gene
(Ym1; also known as Chi313) in BV-2 microglia pretreated with UroA compared to control
upon M2 stimulation with IL-4 plus IL-13 alone (Figure 3B). Notably, RRW treatment did
not alter Ym1 expression. Collectively, these results indicate that UroA promotes M2 po-
larization in microglia similar to other macrophage-like cells, which may be a unique
mechanism by which UroA, but not red raspberry polyphenols, mediates neuroprotective
effects.
Figure 3. Urolithin A attenuated inducible nitric oxide synthase expression and promoted M2 mi-
croglial polarization. (A) BV-2 cells treated with LPS (500 ng/mL) for 3 h followed by ATP (2.5
mM) for 30 min with or without whole red raspberry polyphenols (10 µg/mL) or urolithin A (10
µM). Gene expression levels measured by RT-qPCR for iNos. (B) BV-2 cells treated with IL-4 (100
ng/mL) and IL-13 (10 ng/mL) for 24 h with or without whole red raspberry polyphenols (10
µg/mL) or urolithin A (10 µM). Gene expression levels measured by RT-qPCR for Ym1. All values
Figure 3. Urolithin A attenuated inducible nitric oxide synthase expression and promoted M2microglial polarization. (A) BV-2 cells treated with LPS (500 ng/mL) for 3 h followed by ATP(2.5 mM) for 30 min with or without whole red raspberry polyphenols (10 µg/mL) or urolithin A(10 µM). Gene expression levels measured by RT-qPCR for iNos. (B) BV-2 cells treated with IL-4 (100ng/mL) and IL-13 (10 ng/mL) for 24 h with or without whole red raspberry polyphenols (10 µg/mL)or urolithin A (10 µM). Gene expression levels measured by RT-qPCR for Ym1. All values arerepresented as the mean ± SEM. Treatments with different letters indicate significant differences byone-way ANOVA, p < 0.05. IL = interleukin; LPS = lipopolysaccharide; RRW = whole red raspberrypolyphenols; UroA = urolithin A.
Previously, we demonstrated that UroA induces M2 macrophage polarization in BMDM [26].We therefore tested whether UroA could similarly induce M2 polarization in microglia. Weobserved a significant increase in the expression of the Chitinase-like 3 gene (Ym1; also knownas Chi313) in BV-2 microglia pretreated with UroA compared to control upon M2 stimulationwith IL-4 plus IL-13 alone (Figure 3B). Notably, RRW treatment did not alter Ym1 expression.Collectively, these results indicate that UroA promotes M2 polarization in microglia similar toother macrophage-like cells, which may be a unique mechanism by which UroA, but not redraspberry polyphenols, mediates neuroprotective effects.
Int. J. Environ. Res. Public Health 2021, 18, 68 6 of 11
3.4. Urolithin A Decreased Neuroinflammation in a 12 and 24 h LPS Treatment Model
We further investigated the effect of RRW and UroA on attenuating inflammationusing a longer exposure to LPS for 12 or 24 h. After 12 h of LPS treatment, we observed asignificant increase in the expression of Il1b, Il6, and Tnf compared to untreated microglia(Figure 4A). Treatment with LPS with RRW significantly increased Il1b and Il6 expressionbut decreased Tnf expression compared to LPS alone. Notably, treatment with LPS withUroA significantly decreased Il1b, Il6, and Tnf (Figure 4A). We further assessed the effectsof RRW and UroA on pro-inflammatory cytokine gene expression in microglia following alonger exposure to LPS for 24 h. Similar to a 12 h LPS stimulation, we observed significantincreases in Il1b, Il6, and Tnf after LPS treatment for 24 h, which were all attenuated withUroA treatment (Figure 4B). However, unlike the patterns observed after the 12 h treatment,RRW decreased expression of Il6 and Tnf and had no significant effect on Il1b expressioncompared to microglia treated with LPS alone for 24 h (Figure 4B). Taken together, theseresults suggest UroA, but not RRW, exerts neuroprotective effects during 12 and 24 hLPS exposure.
Int. J. Environ. Res. Public Health 2021, 18, x FOR PEER REVIEW 6 of 11
are represented as the mean ± SEM. Treatments with different letters indicate significant differ-
ences by one-way ANOVA, p < 0.05. IL = interleukin; LPS = lipopolysaccharide; RRW = whole red
raspberry polyphenols; UroA = urolithin A.
3.4. Urolithin A Decreased Neuroinflammation in a 12 and 24 h LPS Treatment Model
We further investigated the effect of RRW and UroA on attenuating inflammation
using a longer exposure to LPS for 12 or 24 h. After 12 h of LPS treatment, we observed a
significant increase in the expression of Il1b, Il6, and Tnf compared to untreated microglia
(Figure 4A). Treatment with LPS with RRW significantly increased Il1b and Il6 expression
but decreased Tnf expression compared to LPS alone. Notably, treatment with LPS with
UroA significantly decreased Il1b, Il6, and Tnf (Figure 4A). We further assessed the effects
of RRW and UroA on pro-inflammatory cytokine gene expression in microglia following
a longer exposure to LPS for 24 h. Similar to a 12 h LPS stimulation, we observed signifi-
cant increases in Il1b, Il6, and Tnf after LPS treatment for 24 h, which were all attenuated
with UroA treatment (Figure 4B). However, unlike the patterns observed after the 12 h
treatment, RRW decreased expression of Il6 and Tnf and had no significant effect on Il1b
expression compared to microglia treated with LPS alone for 24 h (Figure 4B). Taken to-
gether, these results suggest UroA, but not RRW, exerts neuroprotective effects during 12
and 24 h LPS exposure.
Figure 4. Urolithin A, but not whole red raspberry polyphenols, attenuated pro-inflammatory
cytokine gene expression upon 12 and 24 h LPS treatment. (A) BV-2 cells treated with LPS (500
ng/mL) for 12 h with or without whole red raspberry polyphenols (10 µg/mL) or urolithin A (10
µM). Gene expression for Il1b, Il6, and Tnf by RT-qPCR. (B) BV-2 cells treated with LPS (500
ng/mL) for 24 h with or without whole red raspberry polyphenols (10 µg/mL) or urolithin A (10
µM). Gene expression for Il1b, Il6, and Tnf by RT-qPCR. All values represented as the mean ± SEM.
Treatments with different letters indicate significant differences by one-way ANOVA, p < 0.05. LPS
= lipopolysaccharide; RRW = whole red raspberry polyphenols; UroA = urolithin A.
Figure 4. Urolithin A, but not whole red raspberry polyphenols, attenuated pro-inflammatorycytokine gene expression upon 12 and 24 h LPS treatment. (A) BV-2 cells treated with LPS (500 ng/mL)for 12 h with or without whole red raspberry polyphenols (10 µg/mL) or urolithin A (10 µM). Geneexpression for Il1b, Il6, and Tnf by RT-qPCR. (B) BV-2 cells treated with LPS (500 ng/mL) for 24 h withor without whole red raspberry polyphenols (10 µg/mL) or urolithin A (10 µM). Gene expression forIl1b, Il6, and Tnf by RT-qPCR. All values represented as the mean ± SEM. Treatments with differentletters indicate significant differences by one-way ANOVA, p < 0.05. LPS = lipopolysaccharide;RRW = whole red raspberry polyphenols; UroA = urolithin A.
3.5. Urolithin A Regulated the JNK Signaling Pathway upon 12 and 24 h LPS Treatment
Because UroA uniquely limited pro-inflammatory cytokine gene expression in mi-croglia, we subsequently investigated the ability of UroA to downregulate the JNK/c-Junpathways during 12 and 24 h LPS exposure. At both timepoints, LPS treatment significantlyincreased phosphorylation of JNK compared to untreated cells but had no effect on the p-c-Jun pathway (Figure 5). After 12 h, UroA decreased p-JNK protein expression in microglia
Int. J. Environ. Res. Public Health 2021, 18, 68 7 of 11
compared to treatment with LPS alone (Figure 5A). Consistent with proinflammatory geneexpression, RRW treatment had no effect on p-JNK under these conditions (Figure 5A).
Int. J. Environ. Res. Public Health 2021, 18, x FOR PEER REVIEW 7 of 11
3.5. Urolithin A Regulated the JNK Signaling Pathway upon 12 and 24 h LPS Treatment
Because UroA uniquely limited pro-inflammatory cytokine gene expression in mi-
croglia, we subsequently investigated the ability of UroA to downregulate the JNK/c-Jun
pathways during 12 and 24 h LPS exposure. At both timepoints, LPS treatment signifi-
cantly increased phosphorylation of JNK compared to untreated cells but had no effect on
the p-c-Jun pathway (Figure 5). After 12 h, UroA decreased p-JNK protein expression in
microglia compared to treatment with LPS alone (Figure 5A). Consistent with proinflam-
matory gene expression, RRW treatment had no effect on p-JNK under these conditions
(Figure 5A).
Similar to the 12 h LPS exposure, UroA, but not RRW, down-regulated the JNK path-
way in microglia after 24 h treatment of LPS (Figure 5B). Of note, we observed ~67% less
p-JNK protein expression at 24 h compared to 12 h for all treatments (Figure 5B versus
4B). In total, these results demonstrate that UroA, but not RRW polyphenols, downregu-
late the JNK signaling pathway in response to 12 and 24 h LPS treatment.
Figure 5. Urolithin A, but not whole red raspberry polyphenols, regulated the JNK signaling pathway upon 12 and 24 h
LPS treatment. (A) BV-2 cells treated with LPS (500 ng/mL) for 12 h with or without whole red raspberry polyphenols (10
µg/mL) and urolithin A (10 µM). Protein expression from cell lysates for p-JNK, t-JNK, p-c-Jun, c-Jun by Western blot
analysis. (B) BV-2 cells treated with LPS (500 ng/mL) for 24 h with or without whole red raspberry polyphenols (10 µg/mL)
and urolithin A (10 µM). Protein expression from cell lysates for p-JNK, t-JNK, p-c-Jun, and c-Jun by Western blot analysis.
Relative protein intensities normalized to total-JNK or c-Jun by LICOR Image Studio Lite (right). All values represented
as the mean ± SEM. Treatments with different letters indicate significant differences by one-way ANOVA, p < 0.05. Ctrl =
control; N and N.T. = no treatment; W and RRW = whole red raspberry polyphenols; U and UroA = urolithin A.
4. Discussion
The current study was designed to investigate whether whole red raspberry poly-
phenols (RRW) or UroA, the gut-derived metabolite from EA, could attenuate neuroin-
flammation in BV-2 microglia. We found that both UroA and RRW decreased pro-inflam-
matory cytokine expression and JNK/c-Jun pathway activation in response to the 3 h
(LPSplus ATP) induced inflammation. UroA also limited pro-inflammatory responses
during the 12 and 24 h induced neuroinflammation by LPS; however, RRW had neutral
or only minor effects. Moreover, we observed that both UroA and RRW reduced iNos lev-
els, but only UroA promoted Ym1 expression for neuroprotection. Together, these results
indicate a differential temporal regulation of neuroinflammation for RRW and UroA.
Figure 5. Urolithin A, but not whole red raspberry polyphenols, regulated the JNK signaling pathway upon 12 and 24 hLPS treatment. (A) BV-2 cells treated with LPS (500 ng/mL) for 12 h with or without whole red raspberry polyphenols(10 µg/mL) and urolithin A (10 µM). Protein expression from cell lysates for p-JNK, t-JNK, p-c-Jun, c-Jun by Western blotanalysis. (B) BV-2 cells treated with LPS (500 ng/mL) for 24 h with or without whole red raspberry polyphenols (10 µg/mL)and urolithin A (10 µM). Protein expression from cell lysates for p-JNK, t-JNK, p-c-Jun, and c-Jun by Western blot analysis.Relative protein intensities normalized to total-JNK or c-Jun by LICOR Image Studio Lite (right). All values represented as themean ± SEM. Treatments with different letters indicate significant differences by one-way ANOVA, p < 0.05. Ctrl = control; N andN.T. = no treatment; W and RRW = whole red raspberry polyphenols; U and UroA = urolithin A.
Similar to the 12 h LPS exposure, UroA, but not RRW, down-regulated the JNKpathway in microglia after 24 h treatment of LPS (Figure 5B). Of note, we observed ~67%less p-JNK protein expression at 24 h compared to 12 h for all treatments (Figure 5B versus4B). In total, these results demonstrate that UroA, but not RRW polyphenols, downregulatethe JNK signaling pathway in response to 12 and 24 h LPS treatment.
4. Discussion
The current study was designed to investigate whether whole red raspberry polyphe-nols (RRW) or UroA, the gut-derived metabolite from EA, could attenuate neuroinflamma-tion in BV-2 microglia. We found that both UroA and RRW decreased pro-inflammatorycytokine expression and JNK/c-Jun pathway activation in response to the 3 h (LPSplusATP) induced inflammation. UroA also limited pro-inflammatory responses during the 12and 24 h induced neuroinflammation by LPS; however, RRW had neutral or only minoreffects. Moreover, we observed that both UroA and RRW reduced iNos levels, but onlyUroA promoted Ym1 expression for neuroprotection. Together, these results indicate adifferential temporal regulation of neuroinflammation for RRW and UroA.
By using a whole food approach in the present study, we are able to directly comparethe bioactivity of whole red raspberry polyphenols to that of their selective gut metaboliteUroA against neuroinflammation. In recent years, interest in the ability of UroA to provideneuroprotection has increased considerably because this metabolite crosses the blood brainbarrier [11]. Additionally, oral administration of the synthetic gut-metabolite, UroA, or itsprecursor EA, has been shown to be neuroprotective in mice [27]. However, determin-
Int. J. Environ. Res. Public Health 2021, 18, 68 8 of 11
ing whether polyphenols from red raspberries, which consist of ellagitannins as well asanthocyanins, confer this same level of protection as the gut metabolites remains unclear.
Our study uniquely evaluated a polyphenolic extract from whole red raspberriesthat was stripped of any sugars and fibers capable of conferring confounding biologicalactivities. Moreover, our extract contained all of the major polyphenols found in wholered raspberries, including quercetin, myricetin, EA, and anthocyanins, which enabled usto test the neuroprotective effects of whole fruit polyphenols rather than focusing on aconcentrated form of EA, as has been studied previously [21]. Although RRW providedrobust protection against neuroinflammation in LPS/ATP-stimulated microglia, RRW wasonly partly effective in reducing pro-inflammatory cytokine expression during 12 and 24 hLPS exposure, and it had no significant impact on JNK signaling. This result stands incontrast to observations made using a N9 murine microglial model, where a post in vitrodigestion model of raspberries rich in ellagitannin and EA provided neuroprotection forup to 24 h post-LPS stimulation by suppressing the MAPK/NFAT/NF-kB signaling path-ways [10]. In another study, EA pre-treatment decreased TNF-k and NO production fromBV-2 microglia by attenuating MyD88/NF-kB and p38/Erk/JNK signaling pathways afterstimulation with a high dose of LPS (1 ug/mL) for 24 h [28,29]. The inconsistent potency ofour whole red raspberry polyphenol extract with respect to regulating neuroinflammatoryresponses may stem from its composition, as it contains not only free EA but also significantamounts of anthocyanins and epicatechins. Further studies evaluating the contribution ofthese additional polyphenols to neuroprotection are clearly warranted.
Although whole fruit polyphenols provided limited benefits to microglia, we observeda strong and robust neuroprotective effect for UroA, the gut-derived metabolite of RRW,during both 3 h and 12 and 24 h inflammatory conditions. Consistent with our results,others have also shown that UroA attenuates inflammation in BV-2 microglia stimulatedwith LPS for 24 h [17] and regulates pro-inflammatory cytokine expression and MAPKsignaling pathways, including the ERK/p38 signaling pathway [18]. However, the effectsof UroA and RRW following induction of acute neuroinflammation with ATP and LPSrepresentative of ischemia [30,31] have not been investigated. Extracellular ATP is aDAMP leaked from injured brain cells that promotes neuroinflammation. ATP mediatesinflammasome activation and oxidative stress in microglia by acting on purinergic receptorsto promote expression of pro-inflammatory cytokines in addition to activating the P2X7receptor and downstream JNK signaling pathways [32]. ATP is also able to activateMAPK signaling pathways in BV-2 microglia [33]. We observed that both RRW andUroA suppressed 3 h LPS+ATP-induced inflammation driven by LPS plus ATP, possiblyvia antioxidant activities. Further studies are encouraged to understand the mechanismsunderlying why RRW and its gut-derived metabolite, UroA, decrease inflammation acutely.
M2 microglia are thought to provide neuroprotective effects following neuroinflam-mation or associated damage. However, under chronic inflammatory conditions, microgliamay be unable to maintain an M2 polarized state [3]. Previously, our laboratory showedthat UroA was able to promote M2 macrophage polarization in BMDM due to its ability topromote mitochondrial biogenesis [26]. Several studies suggest M2 microglial polarizationis associated with mitochondrial flux rather than glycolytic flux, the latter of which isprominent in BV-2 and primary murine microglia stimulated with LPS [34]. Other workhas shown that mitochondrial uncoupling protein-2 (UCP2) promotes M2 microglial po-larization and prevents neuroinflammation [35]. Moreover, activation of M2 polarizationhas been shown to suppress microglial pro-inflammatory cytokine production and provideneuroprotection [36], which is consistent with our findings. Collectively, these reportssupport the concept that UroA may promote M2 microglial polarization via regulation ofmitochondrial flux.
As neurodegenerative diseases continue to rise, investigating potential therapeuticsthat target pathways of neuroinflammation are imperative. Our findings and those ofothers support the potential for UroA to provide neuroprotective effects. Some studies havefound that the gut microbiome may be crucial in converting EA-rich foods into bioactive
Int. J. Environ. Res. Public Health 2021, 18, 68 9 of 11
urolithins [37–39], while others have evaluated direct oral administration of UroA to healthyhuman adults [40]. However, it remains to be investigated whether these approachesprovide neuroprotective effects to populations at risk of developing neurodegenerativediseases. Mechanistically, UroA and, to a lesser extent, RRW may provide neuroprotectionby regulating the JNK signaling pathway. In ischemia, microglial activation upregulatesJNK signaling pathways and subsequently increases the secretion of pro-inflammatorycytokines [41]. By suppressing JNK and NF-kB downstream signaling with a JNK-In-8 JNKinhibitor, researchers have found that neuroinflammation is inhibited and neurologicalfunction is improved post-ischemic brain injury [41]. Perhaps nanotechnology-baseddelivery vehicles aimed at improving the bioavailability of EA and/or urolithins [42] can,one day, be used to target JNK/NF-kB signaling and provide neuroprotective benefits.
5. Conclusions
Our study demonstrated the differential impact of red raspberry polyphenols andtheir gut-derived metabolite of UroA on neuroprotection in BV-2 microglia. UroA was ableto attenuate pro-inflammatory cytokine gene expression during both 3 h and 12 and 24 hLPS-induced neuroinflammation, in part, by suppressing the JNK signaling pathway andpossibly promoting M2 macrophage polarization. Moreover, RRW suppressed neuroinflam-mation following 3 h LPS/ATP stimulation, but lost its ability to provide neuroprotectionin a 12 and 24 h induced inflammatory setting.
Supplementary Materials: The following are available online at https://www.mdpi.com/1660-4601/18/1/68/s1, Figure S1. LPS with ATP treatment of BV-2 cells did not promote an inflammasomeresponse, Table S1. Phenolic composition of RR extracts, Table S2. Primer sequences for qPCR,Table S3. List of antibodies used in Western blotting.
Author Contributions: Conceptualization, A.M.T.; methodology, A.M.T., A.E.R.-T., S.C.; data acqui-sition, investigation, and curation: A.M.T., M.A., L.P., S.S., A.E.R.-T., S.C.; writing—original draftpreparation, A.M.T. and D.W.; writing—revision: A.M.T., V.C., A.E.R.-T., S.C.; final approval forpublished work: A.M.T., A.E.R.-T., S.C.; funding acquisition, V.C., A.E.R.-T., S.C. All authors haveread and agreed to the published version of this manuscript.
Funding: This work was supported by a USDA-NIFA grant awarded to S.C. and A.E.R.-T. (2017-67017-26781). A.M.T. is supported by a USDA NIFA Predoctoral Fellowship (2019-67011-29518).
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: All data is available based on “MDPI Research Data Policies” athttps://www.mdpi.com/ethics.
Acknowledgments: Equipment was provided by the Nebraska Food for Health Center at the Uni-versity of Nebraska, Lincoln.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
ATP adenosine triphosphate;BMDM bone marrow-derived macrophages;DAMPs damage-associated molecular patterns;EA ellagic acid;ET ellagitannin;JNK c-Jun N-terminal kinases;LPS lipopolysaccharide;RRW whole red raspberry polyphenols;UroA urolithin A
Int. J. Environ. Res. Public Health 2021, 18, 68 10 of 11
References1. Collaborators, G.B.D.N. Global, regional, and national burden of neurological disorders, 1990–2016: A systematic analysis for the
Global Burden of Disease Study 2016. Lancet Neurol. 2019, 18, 459–480. [CrossRef]2. Rodriguez-Gomez, J.A.; Kavanagh, E.; Engskog-Vlachos, P.; Engskog, M.K.R.; Herrera, A.J.; Espinosa-Oliva, A.M.; Joseph, B.;
Hajji, N.; Venero, J.L.; Burguillos, M.A. Microglia: Agents of the CNS Pro-Inflammatory Response. Cells 2020, 9. [CrossRef][PubMed]
3. Cherry, J.D.; Olschowka, J.A.; O’Banion, M.K. Neuroinflammation and M2 microglia: The good, the bad, and the inflamed.J. Neuroinflammation 2014, 11, 98. [CrossRef] [PubMed]
4. Burton-Freeman, B.M.; Sandhu, A.K.; Edirisinghe, I. Red Raspberries and Their Bioactive Polyphenols: Cardiometabolic andNeuronal Health Links. Adv. Nutr. 2016, 7, 44–65. [CrossRef]
5. Kang, I.; Buckner, T.; Shay, N.F.; Gu, L.; Chung, S. Improvements in Metabolic Health with Consumption of Ellagic Acid andSubsequent Conversion into Urolithins: Evidence and Mechanisms. Adv. Nutr. 2016, 7, 961–972. [CrossRef]
6. Marzolo, G. Raspberries. Available online: https://www.agmrc.org/commodities-products/fruits/raspberries (accessed on2 December 2020).
7. Cerdá, B.; Periago, P.; Espín, J.C.; Tomás-Barberán, F.A. Identification of Urolithin A as a Metabolite Produced by Human ColonMicroflora from Ellagic Acid and Related Compounds. J. Agric. Food Chem. 2005, 53, 5571–5576. [CrossRef]
8. Espin, J.C.; Larrosa, M.; Garcia-Conesa, M.T.; Tomas-Barberan, F. Biological significance of urolithins, the gut microbial ellagicAcid-derived metabolites: The evidence so far. Evid. Based Complement. Altern. Med. 2013, 2013, 270418. [CrossRef]
9. Kujawska, M.; Jourdes, M.; Kurpik, M.; Szulc, M.; Szaefer, H.; Chmielarz, P.; Kreiner, G.; Krajka-Kuzniak, V.; Mikolajczak, P.L.;Teissedre, P.L.; et al. Neuroprotective Effects of Pomegranate Juice against Parkinson’s Disease and Presence of Ellagitannins-Derived Metabolite-Urolithin A-In the Brain. Int. J. Mol. Sci. 2019, 21. [CrossRef]
10. Garcia, G.; Nanni, S.; Figueira, I.; Ivanov, I.; McDougall, G.J.; Stewart, D.; Ferreira, R.B.; Pinto, P.; Silva, R.F.; Brites, D.; et al.Bioaccessible (poly)phenol metabolites from raspberry protect neural cells from oxidative stress and attenuate microglia activation.Food Chem. 2017, 215, 274–283. [CrossRef]
11. Yuan, T.; Ma, H.; Liu, W.; Niesen, D.B.; Shah, N.; Crews, R.; Rose, K.N.; Vattem, D.A.; Seeram, N.P. Pomegranate’s Neuropro-tective Effects against Alzheimer’s Disease Are Mediated by Urolithins, Its Ellagitannin-Gut Microbial Derived Metabolites.ACS Chem. Neurosci. 2016, 7, 26–33. [CrossRef]
12. Avila-Galvez, M.A.; Gimenez-Bastida, J.A.; Gonzalez-Sarrias, A.; Espin, J.C. Tissue deconjugation of urolithin A glucuronide tofree urolithin A in systemic inflammation. Food Funct. 2019, 10, 3135–3141. [CrossRef] [PubMed]
13. Ding, Y.; Zhang, B.; Zhou, K.; Chen, M.; Wang, M.; Jia, Y.; Song, Y.; Li, Y.; Wen, A. Dietary ellagic acid improves oxidant-inducedendothelial dysfunction and atherosclerosis: Role of Nrf2 activation. Int. J. Cardiol. 2014, 175, 508–514. [CrossRef] [PubMed]
14. Kang, I.; Espín, J.C.; Carr, T.P.; Tomás-Barberán, F.A.; Chung, S. Raspberry seed flour attenuates high-sucrose diet-mediatedhepatic stress and adipose tissue inflammation. J. Nutr. Biochem. 2016, 32, 64–72. [CrossRef] [PubMed]
15. Djedjibegovic, J.; Marjanovic, A.; Panieri, E.; Saso, L. Ellagic Acid-Derived Urolithins as Modulators of Oxidative Stress. Oxid. Med.Cell. Longev. 2020, 2020, 5194508. [CrossRef] [PubMed]
16. Gonzalez-Sarrias, A.; Nunez-Sanchez, M.A.; Tomas-Barberan, F.A.; Espin, J.C. Neuroprotective Effects of Bioavailable Polyphenol-Derived Metabolites against Oxidative Stress-Induced Cytotoxicity in Human Neuroblastoma SH-SY5Y Cells. J. Agric. Food Chem.2017, 65, 752–758. [CrossRef] [PubMed]
17. Velagapudi, R.; Lepiarz, I.; El-Bakoush, A.; Katola, F.O.; Bhatia, H.; Fiebich, B.L.; Olajide, O.A. Induction of Autophagy andActivation of SIRT-1 Deacetylation Mechanisms Mediate Neuroprotection by the Pomegranate Metabolite Urolithin A in BV2Microglia and Differentiated 3D Human Neural Progenitor Cells. Mol. Nutr. Food Res. 2019, 63, e1801237. [CrossRef]
18. Xu, J.; Yuan, C.; Wang, G.; Luo, J.; Ma, H.; Xu, L.; Mu, Y.; Li, Y.; Seeram, N.P.; Huang, X.; et al. Urolithins Attenuate LPS-InducedNeuroinflammation in BV2Microglia via MAPK, Akt, and NF-κB Signaling Pathways. J. Agric. Food Chem. 2018, 66, 571–580.[CrossRef]
19. Bogoyevitch, M.A.; Kobe, B. Uses for JNK: The many and varied substrates of the c-Jun N-terminal kinases. Microbiol. Mol.Biol. Rev. 2006, 70, 1061–1095. [CrossRef]
20. Waetzig, V.; Czeloth, K.; Hidding, U.; Mielke, K.; Kanzow, M.; Brecht, S.; Goetz, M.; Lucius, R.; Herdegen, T.; Hanisch, U.K.c-Jun N-terminal kinases (JNKs) mediate pro-inflammatory actions of microglia. Glia 2005, 50, 235–246. [CrossRef]
21. Fan, R.; You, M.; Toney, A.M.; Kim, J.; Giraud, D.; Xian, Y.; Ye, F.; Gu, L.; Ramer-Tait, A.E.; Chung, S. Red Raspberry PolyphenolsAttenuate High-Fat Diet–Driven Activation of NLRP3 Inflammasome and its Paracrine Suppression of Adipogenesis via HistoneModifications. Mol. Nutr. Food Res. 2020, 64, 1900995. [CrossRef]
22. García-Villalba, R.; Espín, J.C.; Tomás-Barberán, F.A. Chromatographic and spectroscopic characterization of urolithins for theirdetermination in biological samples after the intake of foods containing ellagitannins and ellagic acid. J. Chromatogr. A 2016, 1428,162–175. [CrossRef] [PubMed]
23. Fan, Z.; Liang, Z.; Yang, H.; Pan, Y.; Zheng, Y.; Wang, X. Tenuigenin protects dopaminergic neurons from inflammation viasuppressing NLRP3 inflammasome activation in microglia. J. Neuroinflammation 2017, 14, 256. [CrossRef]
24. Ding, S.L.; Pang, Z.Y.; Chen, X.M.; Li, Z.; Liu, X.X.; Zhai, Q.L.; Huang, J.M.; Ruan, Z.Y. Urolithin a attenuates IL-1beta-inducedinflammatory responses and cartilage degradation via inhibiting the MAPK/NF-kappaB signaling pathways in rat articularchondrocytes. J. Inflamm. (Lond.) 2020, 17, 13. [CrossRef] [PubMed]
Int. J. Environ. Res. Public Health 2021, 18, 68 11 of 11
25. Sonar, S.A.; Lal, G. The iNOS Activity During an Immune Response Controls the CNS Pathology in Experimental AutoimmuneEncephalomyelitis. Front. Immunol. 2019, 10, 710. [CrossRef] [PubMed]
26. Toney, A.M.; Fan, R.; Xian, Y.; Chaidez, V.; Ramer-Tait, A.E.; Chung, S. Urolithin A, a Gut Metabolite, Improves Insulin SensitivityThrough Augmentation of Mitochondrial Function and Biogenesis. Obesity 2019, 27, 612–620. [CrossRef]
27. Gong, Z.; Huang, J.; Xu, B.; Ou, Z.; Zhang, L.; Lin, X.; Ye, X.; Kong, X.; Long, D.; Sun, X.; et al. Urolithin A attenuates memoryimpairment and neuroinflammation in APP/PS1 mice. J. Neuroinflammation 2019, 16, 62. [CrossRef]
28. Ismail, E.N.; Azmi, N.; Jantan, I. Ellagic Acid Protects against Activation of Microglia by Inhibiting MAPKs and NF-κB Signalling.Indian J. Pharm. Educ. Res. 2020, 54, s529–s536. [CrossRef]
29. He, X.-M.; Zhou, Y.-Z.; Sheng, S.; Li, J.-J.; Wang, G.-Q.; Zhang, F. Ellagic Acid Protects Dopamine Neurons via Inhibition ofNLRP3 Inflammasome Activation in Microglia. Oxid. Med. Cell. Longev. 2020, 2020, 2963540. [CrossRef]
30. Fiebich, B.L.; Akter, S.; Akundi, R.S. The two-hit hypothesis for neuroinflammation: Role of exogenous ATP in modulatinginflammation in the brain. Front. Cell Neurosci. 2014, 8, 260. [CrossRef]
31. Espinosa-Garcia, C.; Atif, F.; Yousuf, S.; Sayeed, I.; Neigh, G.N.; Stein, D.G. Progesterone Attenuates Stress-Induced NLRP3Inflammasome Activation and Enhances Autophagy following Ischemic Brain Injury. Int. J. Mol. Sci. 2020, 21. [CrossRef]
32. Sperlagh, B.; Illes, P. Purinergic modulation of microglial cell activation. Purinergic Signal. 2007, 3, 117–127. [CrossRef]33. Hirata, Y.; Nada, Y.; Yamada, Y.; Toyama, T.; Fukunaga, K.; Hwang, G.W.; Noguchi, T.; Matsuzawa, A. Elaidic Acid Potentiates
Extracellular ATP-Induced Apoptosis via the P2X(7)-ROS-ASK1-p38 Axis in Microglial Cell Lines. Biol. Pharm. Bull. 2020, 43,1562–1569. [CrossRef] [PubMed]
34. Orihuela, R.; McPherson, C.A.; Harry, G.J. Microglial M1/M2 polarization and metabolic states. Br. J. Pharmacol. 2016, 173,649–665. [CrossRef] [PubMed]
35. De Simone, R.; Ajmone-Cat, M.A.; Pandolfi, M.; Bernardo, A.; De Nuccio, C.; Minghetti, L.; Visentin, S. The mitochondrialuncoupling protein-2 is a master regulator of both M1 and M2 microglial responses. J. Neurochem. 2015, 135, 147–156. [CrossRef][PubMed]
36. Yan, C.; Ma, Z.; Ma, H.; Li, Q.; Zhai, Q.; Jiang, T.; Zhang, Z.; Wang, Q. Mitochondrial Transplantation Attenuates Brain Dysfunctionin Sepsis by Driving Microglial M2 Polarization. Mol. Neurobiol. 2020, 57, 3875–3890. [CrossRef]
37. Tomás-Barberán, F.A.; García-Villalba, R.; González-Sarrías, A.; Selma, M.V.; Espín, J.C. Ellagic acid metabolism by human gutmicrobiota: Consistent observation of three urolithin phenotypes in intervention trials, independent of food source, age, andhealth status. J. Agric. Food Chem. 2014, 62, 6535–6538. [CrossRef] [PubMed]
38. Selma, M.V.; Beltrán, D.; García-Villalba, R.; Espín, J.C.; Tomás-Barberán, F.A. Description of urolithin production capacity fromellagic acid of two human intestinal Gordonibacter species. Food Funct. 2014, 5, 1779–1784. [CrossRef]
39. Selma, M.V.; Romo-Vaquero, M.; García-Villalba, R.; González-Sarrías, A.; Tomás-Barberán, F.A.; Espín, J.C. The human gutmicrobial ecology associated with overweight and obesity determines ellagic acid metabolism. Food Funct. 2016, 7, 1769–1774.[CrossRef]
40. Andreux, P.A.; Blanco-Bose, W.; Ryu, D.; Burdet, F.; Ibberson, M.; Aebischer, P.; Auwerx, J.; Singh, A.; Rinsch, C. The mitophagyactivator urolithin A is safe and induces a molecular signature of improved mitochondrial and cellular health in humans.Nat. Metab. 2019, 1, 595–603. [CrossRef]
41. Zheng, J.; Dai, Q.; Han, K.; Hong, W.; Jia, D.; Mo, Y.; Lv, Y.; Tang, H.; Fu, H.; Geng, W. JNK-IN-8, a c-Jun N-terminal kinaseinhibitor, improves functional recovery through suppressing neuroinflammation in ischemic stroke. J. Cell Physiol. 2020, 235,2792–2799. [CrossRef]
42. Almuhayawi, M.S.; Ramadan, W.S.; Harakeh, S.; Al Jaouni, S.K.; Bharali, D.J.; Mousa, S.A.; Almuhayawi, S.M. The potential roleof pomegranate and its nano-formulations on cerebral neurons in aluminum chloride induced Alzheimer rat model. Saudi J.Biol. Sci. 2020, 27, 1710–1716. [CrossRef] [PubMed]